The present invention relates to a novel method to detect D. immitis infection in animals, particularly in cats. The present invention also includes novel kits to detect D. immitis infection as well as methods to purify the detection reagent.
The parasitic helminth D. immitis has been known for a long time to infect dogs, thereby leading to heartworm. Recently, it has become clear that D. immitis also infects other animals, such as cats and ferrets, even though such animals are essentially non-adpated hosts for the infection. That is, the parasitic relationship between D. immitis and adapted animals is well adapted, and there are very few clinical signs unless worm burden is very high. In contrast, non-adapted animals, such as cats and ferrets, have a parasitic relationship with D. immitis that is not well adapted, resulting in disease and sometimes death for the animal. Heartworm infection in non-adapted animals such as cats is hard to diagnose as there are a variety of clinical signs, some of which are also associated with other diseases. Heartworm disease may be acute or chronic, and can be associated with dyspnea, coughing and vomiting, lethargy, and/or anorexia. Thus, there is a need for an unambiguous method to detect heartworm in animals.
The life cycle of D. immitis is complex in all animals it infects, and the organism is difficult to detect, particularly prior to adult worm maturation. Detection is particularly difficult in non-adapted hosts where the worm burden is very low. Cats, for example, harbor, on average, only two to three worms, making detection of D. immitis-specific antigens or antibodies difficult.
Sexually mature adults, after mating, produce microfilariae which traverse capillary beds and circulate in the vascular system of the host. One method of demonstrating infection in a dog, for example, is to detect the circulating microfilariae. Another method is to detect D. immitis circulating parasite antigens in the blood; these antigens are associated with adult female worms and microfilariae (see, for example, U.S. Pat. No. 4,839,275, issued Jun. 13, 1989, by Weil). In a non-adapted host, however, D. immitis infection often results in the maturation of only a single worm, in which case there is no opportunity for reproduction, and eggs and microfilariae are not produced. In addition, the single worm is often a male worm.
If an infected animal is maintained in an insect-free environment, the life cycle of the parasite cannot progress. However, when microfilariae are ingested by the female mosquito during blood feeding on an infected animal, subsequent development of the microfilariae into larvae occurs in the mosquito. The microfilariae go through two larval stages (L1 and L2) and finally become mature third stage larvae (L3) which can then be transmitted back to a host animal through the bite of the mosquito. It is this L3 stage, therefore, that accounts for the initial infection. As early as three days after infection, the L3 molt to the fourth larval (L4) stage, and subsequently to the fifth stage, or immature adults. The immature adults migrate to the heart and pulmonary arteries, where they mature and reproduce, thus producing the microfilariae in the blood. xe2x80x9cOccultxe2x80x9d infection with heartworm in a host is defined as that wherein no microfilariae can be detected, but the existence of the adult heartworms can be determined by other methods.
Another method to detect heartworm is the use of crude preparations; see, for example, U.S. Pat. No. 4,657,850, issued Apr. 14, 1987, by Grieve. These assays, however, lack desired sensitivity and specificity, particularly to detect infection in a non-adapted host.
Hong et al, 1995, Proc. Heartworm Symposium, p. 33, reported the cloning of a gene encoding D. immitis antigen DiT33; also see Hong et al, 1994, Abstracts of Amer. Soc. Trop. Med.Hyg. Meeting, p191-192. Hong et al., 1995, ibid, also reported that a recombinant fusion protein consisting of DiT33 linked to maltose binding protein could detect D. immitis infection in dogs at 11 weeks but did not report any use of the protein to detect D. immitis infection in a non-adapted host, such as in a cat or ferret. Previously, several investigators had reported the use of the related protein Onchocerca volvulus Ov33 to detect O. volvulus or D. immitis infection, as well as cloning of the gene encoding O. volvulus Ov33; see, for example, Santiago Mejia et al, 1994, Parasite Immunol 16, 297-303; Ogunrinade et al, 1993, J Clin Microbiol 31, 1741-1745; Lucius et al., 1992, Trop Med Parasitol 43, 139-145; Lucius et al, 1988, J. Exp Med 168, 1199-1204; Lucius et al, 1988, J. Exp Med 167, 1505-1510. A related gene encoding Av33 has been isolated from Acanthocheilonema viteae; see, for example, Willenbucher et al, 1993, Mol. Biochem. Parasitol. 57, 349-351. Once again, there was no mention of the ability of that protein to detect infection in an animal with a low worm burden prior to adult worm maturation.
There remains a need for an accurate and simple method to detect D. immitis infection. Particularly needed is a method that would detect D. immitis infection prior to maturation of larvae into adult heartworms, but would not detect early infections that never develop into full-term infections, i.e., infections that the host immune response is able to prevent from developing into mature worms.
The present invention includes detection methods and kits that detect D. immitis infection prior to maturation of larvae into adult heartworms, but do not detect early infections that do not develop into full-term infections (i.e., infections that do not lead to mature heartworm development).
The present invention includes a method to detect D. immitis in a non-adapted host that includes the steps of: (a) contacting a bodily fluid collected from the host with a formulation comprising an isolated D. immitis Di33 protein under conditions sufficient to form an immunocomplex between Di33 protein and anti-Di33 antibodies; and (b) measuring immunocomplex formation between the Di33 protein and anti-Di33 antibodies, if any, in the fluid, wherein the presence of such an immunocomplex indicates that the host is or has recently been infected with D. immitis. 
The present invention also includes a method to detect D. immitis in a host animal, which includes the steps of: (a) contacting a bodily fluid collected from the animal with a formulation comprising an isolated D. immitis Di33 protein under conditions sufficient to form an immunocomplex between Di33 protein and anti-Di33 IgE antibodies; and (b) immunocomplex formation between the Di33 protein and anti-Di33 IgE antibodies, if any, in the fluid, wherein the presence of such an immunocomplex indicates that the animal is or has recently been infected with D. immitis. 
Also included in the present invention is a method to detect D. immitis infection in a non-adapted host within 10 weeks of infection, the method comprising detecting anti-Di33 antibodies in a bodily fluid collected from the host.
The present invention also includes a method to detect D. immitis in a non-adapted host, that includes the steps of: (a) contacting a bodily fluid collected from the host with a formulation comprising an isolated anti-Di33 antibody under conditions sufficient to form an immunocomplex between the anti-Di33 antibody and D. immitis Di33 protein; and (b) measuring immunocomplex formation between the anti-Di33 antibody and D. immitis Di33 protein, if any, in the fluid, wherein the presence of such an immunocomplex indicates that the host is or recently has been infected with D. immitis. 
One embodiment of the present invention is a kit to detect D. immitis infection that includes an isolated D. immitis Di33 protein and a composition to detect antibodies capable of forming an immunocomplex with the Di33 protein. Another embodiment is a kit to detect D. immitis infection that includes an isolated anti-Di33 antibody and a composition to detect an immunocomplex between the anti-Di33 antibody and D. immitis Di33 protein.
The present invention also includes Di33 proteins, nucleic acid molecules encoding such proteins, as well as recombinant molecules and recombinant cells comprising such nucleic acid molecules, and anti-Di33 antibodies. Examples of Di33 proteins include, but are not limited to, PHIS-PDi33234 and PDi33217. Examples of Di33 nucleic acid molecules include, but are not limited to, nDi33346, nDi33750, nDi33702, nDi33708, and nDi33651. Also included are methods to produce Di33 nucleic acid molecules, Di33 nucleic acid molecule-containing recombinant molecules, Di33 nucleic acid molecule-containing recombinant cells, Di33 proteins and anti-Di33 antibodies.
One embodiment of the present invention is a method to produce an isolated D. immitis Di33 protein that includes the steps of (a) culturing a bacterium transformed with a D. immitis Di33 nucleic acid molecule to produce a D. immitis Di33 protein-containing culture; (b) recovering insoluble material comprising the Di33 protein from the culture; and (c) purifying the Di33 protein from the insoluble material. Also included is a method for purifying a D. immitis Di33 protein comprising recovering Di33 protein from cation exchange chromatography of disrupted insoluble material obtained from a culture of D. immitis Di33 protein-producing recombinant cells.